L9: Generation of Diversity

Question 1

What is the T cell receptor made up of?

Answer 1

2 chains, an alpha chain and a beta chain

Question 2

What is the B cell receptor made up of?

Answer 2

A heavy chain and a light chain
There are 2 light chain genes: kappa and delta (one or the other is included in the BCR expressed by an individual B cell)

Question 3

What processes generate diversity in both B cells and T cells?

Answer 3

1. Multiple copies of each gene segment that makes up the variable joined in different combinations (combinatorial)
2. Addition and subtraction of nucleotides by the recombination process (junctional diversity)
3. Many different combinations of H and L chain V regions (or alpha and beta V regions) that pair to form the antigen binding site (combinatorial)

Question 4

What additional processes add to BCR diversity?

Answer 4

4. Somatic hypermutation occurring in the germinal center

Question 5

How many antigen binding sites do TCRs and BCRs have? What are they made up of?

Answer 5

BCRs: 2 identical antigen binding sites; formed by the heavy chain and the light chain

TCRs: Singular antigen binding site; formed by the alpha and beta chains

Question 6

Where are BCRs expressed?

Answer 6

On surface of B cell or secreted as antibody

Question 7

Why is generation of diversity important?

Answer 7

Survival is dependent upon a wide range of antigen specific receptors, differing in their amino acids sequences at the antigen binding site

But encoding each receptor chain variant in the genome would require more genes than are present in the entire genome

Question 8

What undergoes splicing in TCR and BCR genes?

Answer 8

Both the DNA itself as well as the primary RNA transcript

Question 9

Describe Ig light chain gene construction

Answer 9

Light chain variable region (V_L) is constructed from 2 segments, a variable (V) and joining (J) gene segment; there is a leader peptide (L) that precedes the V gene segment

V and J gene segments in the genomic DNA are joined to form a complete V_L region exon → Light-chain C region (C_L) is encoded in a separate exon and is joined to the V_L exon by splicing of the light-chain RNA to remove the L-to-V and J-to-C introns

Question 10

Describe Ig heavy chain gene construction

Answer 10

Heavy chain V region (V_H) is constructed from 3 gene segments: the diversity (D), V, and J segments

D and J gene segments join → V gene segment joins to the combined DJ sequence → forms a complete V_H exon → During RNA processing of heavy-chain RNA transcript, C-region exons, together w/ leader sequence, are spliced to the V_H domain sequence

Question 11

How many gene segments are there for V, D, and J loci?

Answer 11

Multiple gene segments for each

For instance, for heavy chain there are 45 possible V regions

Question 12

What is intragenic recombination mediated by?

Answer 12

Mediated by the RAG recombinases recognizing RSS (recombination signal sequence)

Question 13

Describe what RAG recombinases do?

Answer 13

RAG recombinase components (RAG-1 and RAG-2) align the segments that will be spliced and excise the intervening DNA including the RSS sequence

Get a signal joint and a coding joint (this gives you VJ region; this is the spliced region that will be expressed)

Rejoining of DNA ends is done by DNA repair enzymes

Question 14

Why does immunodeficiency occur if there is a problem w/ DNA repair enzymes?

Answer 14

Can’t complete DNA repair necessary for T and B cell diversity since can’t resolve the cuts that the RAG enzymes have made

Question 15

Where are RAG-1 and RAG-2 expressed?

Answer 15

Only in developing lymphocytes

since don’t want it recombining genes in random

Question 16

Is ligating at the ends of the signal joint and the coding joint precise?

Answer 16

At the signal joint, DNA ligase joins the blunt ends precisely

At the coding join, ligation occurs imprecisely, thus inducing additional variability y into the variable region

Question 17

TdT

Answer 17

(Terminal deoxytransferase)
Adds N (nontemplated) nucleotides at the coding joints; this is a random process

Question 18

What can aberrant rearrangements lead to?

Answer 18

Chromosomal translocations and malignancy

The unique VDJ rearrangement aids in recognizing the presence of a lymphoid malignancy

Question 19

In B cell development, does H chain or L chain gene rearrangement occur first?

Answer 19

VDH rearrangement of heavy chain occurs first

VJ rearragement of light chain then occurs

Question 20

How do nonproductive rearrangements occur?

Answer 20

Added nucleotides at the junctions often disrupt the reading frame, causing a nonproductive rearrangement in 2/3

Question 21

What happens when nonproductive rearrangements occur?

Answer 21

B cell progenitors that do not produce functional immunoglobulin never mature

Question 22

If rearrangement in unsuccessful, what occurs?

Answer 22

Try again since there are 2 alleles

If get it wrong twice, there is cell death

Once able to express heavy chain protein, the rearrangement for light chain protein begins

Question 23

What do all BCR constant regions start off as?

Answer 23

IgM

IgM and IgD seem to be expressed in early B cells

Question 24

What is expressed on the surface of naive B cells?

Answer 24

IgM and IgD

These cell have not yet undergone class-switching

Question 25

In germline TCR genes, are there D regions?

Answer 25

Alpha-chain locus has V and J

Beta-chain locus has V, D, and J

Question 26

If you have defects in VDJ recombination in either RAG or in DNA repair enzymes, what cells are going to have defects?

Answer 26

Both B and T cells

Question 27

Describe TCR rearrangement compared to BCR rearragement

Answer 27

Occurs by the same mechanisms

RAG-1/2 bind to RSS sequences and mediate DNA breakages

Rejoining is mediated by the same DNA repair enzymes

Addition of P and N nucleotides at joints occurs in the same way

Defects in VDJ recombination affect both T and B cells

Somatic hypermutation does not occur after TCR rearrangement